Cell-type specification that occurs during development and to some extend during adulthood of an animal depends on both quantitative and qualitative differences in gene expression (see, e.g., Lodish et al., Molecular Cell Biology, W.H. Freeman and Company, New York, N.Y., 2000). Certain genes are only expressed in a specific cell type or lineage and are important in cell-type specification. Genes involved in housekeeping tasks or in processes fundamental to all cell types generally are more ubiquitously expressed. Regulation of transcription is a widespread form of gene expression regulation involving interaction between transcription factors and co-factors with gene promoters and the basal transcriptional machinery. Genome or chromosomal remodeling may also contribute to transcriptional regulation.
Transcriptional regulation is an important process in regulating gene expression in stem cells, and plays a critical role in cell fate, i.e., cell specification, cell determination, and cell differentiation. Transcriptional control is maintained in embryonic stem cells (“ESCs”) by several “key regulators”—transcription factors specifically expressed in ESCs but not expressed in differentiated tissues—which include Oct4, Sox2 and Nanog (see, e.g., Cole and Young, Cold Spring Harb. Symp. Quant. Biol., 2008, 73:183-193). Oct4-Sox2 and Nanog work in concert with one another, and often are bound together to the promoter regions upstream from the same set of genes (see, e.g., Loh, Nat. Genetics, 2006, 38:413-440).
Oct4-Sox2 are specifically expressed in undifferentiated ESCs and form a stable heterodimer. Expression of Oct4 is necessary for the maintenance of stem cell pluripotency, and can serve as a stem cell marker. In the absence of Oct4, pluripotent stem cells revert to the trophoblast lineage.
The Oct4-Sox2 binding sites on promoters are typically adjacent to one another. Sox2 typically binds to a “Sox element” with the consensus sequence CATTGTA, and Oct 4 binds to an “Oct element” with the consensus sequence ATGCAAAA. These two motifs may be contiguous in the DNA sequence, and may be present in forward or reverse orientation.
The promoter region of Oct4 has been well characterized (GenBank Accession No. AP000509). The region encompasses −3917 to +55 basepair (bp) relative to the transcription start site (see, e.g., Nordhoff et al., Mammalian Genome, 2001, 12:309-317). The minimal promoter region is within the first 250 bps of the transcription start site, and enhancers and other regulatory elements, such as repressor elements, are further upstream. The full promoter region can drive tissue- and cell-specific expression of a reporter construct containing a gene of interest (see, e.g., Gerrard et al., Stem Cells, 2005; 23:124-133).
Nanog (GenBank Accession No. NT—009714, GenBank: AC006517) expression is driven by the Nanog promoter. This Nanog promoter region encompasses roughly 400 bp (−289 to +117 bp relative to the transcription start site) (see, e.g., Rodda et al., J. Biol. Chem., 2005, 280(26):24731-24737). A region of roughly 200 bps within the Nanog promoter is highly conserved. This conserved region contains a “Sox element” (CATTGTA) and an “Oct element” (ATGCAAAA) adjacent to one another, both in reverse orientation. These elements are binding sites for the Sox2-Oct4 heterodimer.
This promoter region can be used to drive ESC-specific expression of a gene of interest. For example, addition of this promoter region upstream from an eGFP reporter drives expression patterns in ESCs that are identical to endogenous Nanog (see, e.g., Rodda et al., J. Biol. Chem., 2005, 280(26):24731-24737).
Stem cells are self-renewing cells that divide to give rise to daughter cells that can have an identical developmental potential and/or daughter cells with a more restricted (e.g., differentiated) developmental potential (see, e.g., Lodish et al., Molecular Cell Biology, W.H. Freeman and Company, New York, N.Y., 2000). Stem cells can also be found in small numbers in various tissues in the fetal and adult body. Stem cells can be obtained from other sources, for example, the umbilical cord of a newborn baby is a source of blood stem cells. Stem cells are described in terms of their potency—that is how many and how broad are the cell types they are capable of producing (see, e.g., Weiner et al., Methods Mol. Biol., 2008, 438:3-8). Multipotent stem cells are capable of repopulating a defined tissue, whereas pluripotent stem cells are capable of giving rise to all three germ layers-endoderm, mesoderm and ectoderm (see, e.g., Smith et al., J. Cell Physiol., 2009, 220(1):21-9). Pluripotent stem cells, such as ESCs, also have the capability of self-renewal. ESCs are derived from the inner cell mass of the blastocyst.
Recently it has been shown that expression of a cocktail of genes (i.e., c-Myc, Klf4, Oct4, and Sox2) known to be important in the maintenance of the stem cell state in ESCs, can reprogram mature or somatic cells to a cell indistinguishable from an ESC, which is termed an induced pluripotent stem (iPS) cell (see, e.g., Woltjen et al., (2009) Nature, 458:766-770). Both ESCs and iPS cells are capable of being maintained long term in a stem cell state in vitro. Both cell types when injected into mice, give rise to teratomas, tumors containing cells derived from all three germ layers.
In the adult, there are thought to be stem cells residing in each tissue that are capable of repopulating a defined tissue in the course of maintenance and repair (see, e.g., Pekovic et al., J. Anat., 2008, 213(1):5-25). Hematopoietic stem cells (HSCs) reside in the bone marrow and are capable of giving rise to all the cells in the blood and bone marrow, including red blood cells, macrophages and other immune cells (see, e.g., Weissman I L, Annu Rev. Cell Dev. Biol., 2001, 17:387-403). A special type of HSC from blood and bone marrow called “side population” or “SP” is described as CD34-/low, c-Kit+, and Sca-1+ (see, e.g., Jackson et al., (2001) J. Clin. Invest., 107(11): 1395-1402).
Other well defined adult stem cell populations include neural stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, adipose stem cells, olfactory stem cells and skin stem cells. These cells reside in a well defined “niche” environment in vivo that plays a key role in maintaining the stem cell state. Ex vivo culture of adult stem cells usually results in the differentiation of these cells. When harvested from a donor and given to a recipient, these cells are able, under certain conditions, to engraft in the recipient and contribute to the mature tissue (see, e.g., Sensebé et al., Transplantation, 2009, 87(9 Suppl):S49-S53).
Currently there is a demand for a screening system for modulators of cell fate, wherein the screening system is suitable for high throughput screening. The present invention provides such a system.